The main goal of the Phase II project is to build on the success of the Phase I study to develop a cost-effective multiplex serum assay with an expanded biomarker panel for detection of clinically significant forms of prostate cancer (PCa), and perform a larger scale preclinical study of the assay using PharmaSeq's p-Chip-based platform. In the Phase I study of 70 PCa serum samples, we determined that Macrophage Inhibitory Cytokine 1 (MIC-l) has promise for prostate cancer diagnosis and that including serum levels of MIC-l with prostate specific antigen (PSA) improves specificity of PCa diagnosis without compromising the high sensitivity of the PSA test. In the Phase II study, we propose to expand the panel of markers in the PCa assay to include, in addition to MIC-l, Endoglin, Interleukin-8 (IL-8) and Annexin A3. The latter three biomarkers were previously used in a multiplex urine assay, and when combined, were found to significantly outperform the current serum PSA test for PCa diagnosis (a recent study at PharmaSeq and JHUSOM). The benefits of using the four biomarkers will be evaluated in the multiplex serum-based assay at JHUSOM and at PharmaSeq. Silver nanolayer-based metal-enhanced fluorescence was demonstrated to provide an ultrasensitive assay in the Phase I study and will be applied in the Phase II work, if needed. The multiplex assay is adapted to PharmaSeq's innovative platform, the key element of which is the p-Chip, a unique RFID microtransponder, which is capable of transmitting its ID via radio frequency upon being activated by light. The readout is performed on a custom fluidics-based analyzer for p-Chips that simultaneously determines the fluorescence intensity and the p-Chip ID. The following tasks will be performed: 1) final development of assay and instrumentation, 2) assay qualification and evaluation of performance and 3) preclinical study (proving assay utility in PCa diagnostics) using a larger set of samples. The assay results will be then combined with results of PSA analysis of the samples and used to develop a new calculation method utilizing multiple analytes for PCa diagnosis. The new calculation method is aimed at improving specificity of PCa diagnosis while maintaining high sensitivity of the PSA test to minimize the current high false positive rate. The project is expected to provide a major cost and life style benefit to society: this new multiplex diagnostic assay for PCa could earlier identify clinically significant PCa patients requiring intervention, especially when the results are combined with the routine serum PSA test.